Brucella is a zoonotic pathogen for which no safe, effective human vaccine exists. In addition, the mechanisms of Brucella virulence are poorly understood. Our long-term goal is to develop effective vaccine strategies against B. melitensis and use our methodologies to elucidate mechanisms of Brucella pathogenesis for vaccine strategies. Our specific hypothesis is that live pathogenic Brucella express immunomodulation factors during infection that are necessary for a lasting protective immune response by the host. First, we will identify Brucella immunomodulation traits and virulence factors. We will distinguish these traits by: 1) Microarray gene expression analysis of Brucella within infected cells to identify strategic pathogen genes for host immune response. 2) Candidate Brucella virulence genes will be isolated, cloned, and examined to assess interaction with host proteins. 3) Immunomodulation gene knockout/ complementation assays to assess function. Second, we will determine the host adaptive immune response to Brucella infection. We will examine immune regulation by: 1) Host microarray gene expression analysis of infected cells to identify genes central for host immune response. 2) Library-based, virulent Brucella peptide screening to identify significant Brucella T cell antigens for vaccine application. 3) Adoptive transfer of immune cell subsets, cytokine profiling, and microfluorimetry using knock-out as well as human immune system (humanized) mice to elucidate the host response to Brucella pathogenesis. Third, we will develop a safe vaccine conferring effective, lasting immunity to brucellosis. We will define this vaccine as follows: 1) Irradiated Brucella to determine dosage/kill curves where replication is inhibited, but metabolic activity occurs. Bacterial CPU will assay replication and redox activity, /uxtransgene expression, and RTPCR of Brucella gene expression will measure metabolic activity. 2) Irradiated Brucella immunomodulation and immune response for comparison to pathogenic and attenuated vaccine strains through gene expression kinetic profiling. 3) Irradiated Brucella vaccine protection to be measured in the mouse model based on challenge clearance using in vivo imaging and CPU counts implementing established control methods and statistical thresholds of acceptability for anti-Brucella vaccines. Quality and type of immunity will be assessed utilizing microfluorimetry, cell transfer, and expression profiling. These complementing aims will provide definitive understanding of the host-pathogen response in brucellosis and contribute to the challenges facing Brucella vaccine development and design.